Chemical signals from the environment regulate a diverse array of animal behaviors, essential for survival and regulation of social interactions. Most animals have developed a dual olfactory system for the recognition of these chemical signals: the main olfactory system, which receives input from olfactory neurons in the nose, and the accessory olfactory system, which receives input from chemosensory neurons in the vomeronasal organs (VNO). Although considerable information is now available regarding stimulus recognition, signal transduction and odor coding in the olfactory system, little is known about the repertoire of chemical stimuli that are received via the VNO and the transduction pathways they activate. In previous experiments using mice we have shown that compounds in adult male urine activate the production of the second messenger, IP3, in microvillar membranes from VNOs of prepubertal females. Stimulus-induced release of IP3 is dose dependent, sex dependent, tissue-specific and GTP-dependent, indicating that this process is receptor mediated and involves a regulatory G-protein. In this grant application we propose to continue our experiments that use the IP3 responses in murine VNO membranes as a biochemical assay to purify and characterize compounds from male urine that activate the release of IP3 in membrane preparations from female VNOs. We will also evaluate the contributions of Gi2, Gi3, Go, and Gq/11, all present in VNO membranes, in regulating stimulus-dependent production of IP3. The extent to which each of these G-proteins contributes to activation of the IP3 transduction pathway in the vomeronasal organ will be evaluated by a combination of Bordetella pertussis toxin mediated ADP-ribosylation studies and experiments using site-directed antibodies against specific G-protein alpha subunits as functional inhibitors. We will delineate the mechanisms by which stimuli are transduced into neural messages, by examining by immunohistochemical and biochemical procedures the expression of IP3 receptor isoforms in the vomeronasal organ. Finally we will determine if G-protein Receptor Kinases (GRK2 and GRK3) are selectively expressed in the vomeronasal organ and determine translocation of these GRKs upon pheromonal stimulation. These studies will increase our understanding of the diversity of VNO stimuli and their modes of action on the VNO and in the long term, contribute to shedding light on the possible function of the VNO in humans and the possible impact on VNO stimuli on human physiology and behavior.